Peptide antagonists of substance P

ABSTRACT

Undecapeptides retaining the amino acids in positions 2, 3, 4, 5, 6, 8 and 10 of substance P (Arg 1  -Pro 2  -Lys 3  -Pro 4  -Gln 5  -Gln 6  -Phe 7  -Phe 8  -Gly 9  -Leu 10  -Met 11  -NH 2 ), but having substitutions in positions 1, 7, 9 and 11 of substance P have been discovered to have high antagonistic activity to block substance P in biological systems. Exemplifying these potent antagonists is D-Arg 1 ,D-Trp 7 ,D-Trp 9  -Leu 11  -Substance P, which is an effective inhibitor and has high potency. These antagonists of substance P are useful to elucidate some biological mechanisms of substance P, and to treat inflammatory responses in the eye for medical practice in ophthalmology, and to be novel analgesic agents for medical applications.

BACKGROUND OF THE INVENTION

The sequence-biological activities of the luteinizing hormone releasinghormone (LHRH) (Glp-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) andsubstance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂)differ in an important structural aspect. No truncated analog of LHRH isknown which has activity equal to that of LHRH. Although LHRH is adecapeptide and SP is an undecapeptide, the removal of the N-terminalfour or five amino acids of SP gives truncated hexa- and heptapeptideswhich show biological activities essentially equivalent ot that of SPitself. For example, the studies of Yanaihara et al. ["Substance P NobelSymposium 37", U.S. von Euler and B. Pernow, eds., Raven Press, New York(1977) p. 27] exemplify data on sequence-activity of truncated analogsof SP, which showed that the hexapeptide, Glp-Phe-Phe-Gly-Leu-Met, andthe heptapeptide, Glp-Gln-Phe-Phe-Gly-Leu-Met-NH₂, showed 2.0 and 1.7relative activities, respectively, in comparison with SP as 1.

It is generally believed in the field of SP that the N-terminal Arg¹-Pro² -Lys³ -Pro⁴ - or Arg¹ -Pro² -Lys³ -Pro⁴ -Gln⁵ - of SP are notneeded for biological activity and such statements imply the conceptthat these N-terminals four or five amino acids have no biological rolewhatsoever. This concept may not be entirely correct, because it ispossible that these four or five N-terminal amino acids of SP have arole in binding at one or more receptors, particularly when oneconsiders the strong functionality of Arg¹ and Lys³ in these units.Also, the presence of Pro² and Pro⁴ indicate a unique conformationalcontribution from the N-terminal portion of SP. Therefore, the presenceof Arg¹ -Pro² -Lys³ -Pro⁴ - and Arg¹ -Pro² -Lys³ -Pro⁴ -Gln⁵ - shouldnot be summarily dismissed from consideration of some nature of abiological role.

Doubtless, peptide chemists, in general, were attracted to synthesizetruncated analogs of SP not only because the N-terminal moiety does notcontribute to agonist mechanisms, but because truncated analogs can bemore quickly synthesized, and at less expense, and probably be purifiedto a high state of purity more readily than the correspondingundecapeptides.

We have investigated truncated analogs of SP as antagonists incomparison with the corresponding undecapeptides. In doing so, we havesurprisingly discovered certain undecapeptides which have higherantagonistic activity than any previously known analog of SP. We havealso discovered that a certain truncated analog has substantially lessantagonistic activity than that of the corresponding undecapeptide. Thisfinding that a potent undecapeptide has substantially more antagonisticactivity than a corresponding truncated peptide is not in agreement withthe comparable agonistic activities of truncated analogs of substance P.

THE INVENTION

It has been discovered, in accordance with the present invention, thatundecapeptides having Pro² -Lys³ -Pro⁴ -Gln⁵ -Gln⁶ -Phe⁸ -Leu¹⁰ as doessubstance P, but have substitutions in positions 1, 7, 9 and 11, such as[D-Arg¹,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-SP have exceptional antagonistic activityand greater than that of any previously described analog of substance P;in addition, it has been surprisingly discovered that the truncatedanalog, which is [GlP⁵,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-SP₅₋₁₁, of thisexceptionally potent antagonist is substantially less active than itscorresponding undecapeptide, [D-Arg¹,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-SP.

The exceptional antagonistic activity of [D-Arg¹,D-Trp⁷,D-Trp⁹,Leu¹¹]-SP makes this peptide very useful, over and above previous peptides,such as [D-Pro²,D-Trp⁷,D-Trp⁹ ]-SP. [D-Arg¹,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-SP issubstantially more potent than [D-Pro²,D-Trp⁷,D-Trp⁹ ]-SP, and is moreuseful to alleviate inflammatory symptoms in the eye, according tomedical practice is ophthalmology, particularly since topicalapplication is sufficient to reduce inflammation.

This remarkably attractive antagonist was synthesized by solid-phaseexperimentation. The deprotection and cleavage of the undecapeptide fromthe resin was accomplished by hydrogen fluoride and the resultingpeptidic material was purified to yield the pure[D-Arg¹,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-SP.

GENERAL METHODS

The acquisition is protected amino acids, the synthesis of the peptides,the cleavage of the peptides from the resin, were conducted as describedfor the previous analogs and antagonists of substance P [K. Folkers, J.Horig, S. Rosell and U. Bjorkroth, Acta Physiol. Scand. 111, 505(1981)].

The modified purification of the crude peptides, as carried out for thepeptides described herein, was conducted as follows. Samples of about200 mg of the crude peptides were applied to a column of Sephadex G-25(100×2.5 cm) which had been equilibrated with 12% acetic acid, and thenchromatography was carried out with the same solvent. Fractions of 10 mlwere collected. The peptides were detected by spotting samples of theindividual fractions as silica gel plates and conducting thechromatography with n-BuOH:HOAc:H₂ O=4:1:2. The chromatography wascarried out with this same solvent system, and fractions of 4 ml werecollected. The desired peptides, in general, were found in fractions30-40. Those fractions which contained the pure or nearly pure peptidewere collected and lyophilized. If the desired peptide were notsufficiently pure, it was again purified over silica gel using the samesolvent system. The yields of the peptides were 20-50%. Purity was moreimportant than the yield, and ranged from 90 to 98%, which wasacceptable for the first sample for the assay and its error. The timefor achievement of purity was balanced with the time of synthesis andassay and the achievement of higher potency of antagonism which was theimportant goal. The peptidic impurities were not a deterrent toward thegoal. High performance liquid chromatography was conducted on a WatersLiquid Chromatograph equipped with a Waters 660 solvent programmer. Thesamples were chromatographed on a μ-Bondapak C₁₈ column (10μ), 3.9×300mm. For elution of the peptides, a linear gradient from 20-100% of asolvent system was used during 25 min. The solvent system consisted of70% of CH₃ CN and 30% 0.1M K H₂ PO₄ buffer, pH 3. The flow rate was 2.0ml/min. and 10 μl of a 0.1% solution of the peptide was injected. Theeluted peptide was detected by its UV absorbance at 206 nm.

EXEMPLARY SYNTHESIS OF [D-Arg¹,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-SP

The protected amino acids were purchased from Peninsula Laboratories,Inc., San Carlos, Calif. α-Amino functions were protected by theBOC-group. Side-chain functions were protected by o-Cl-Z for α-aminofunction of Lys, Tos for Arg. The benzhydrylamine hydrochloride resinwas obtained from Beckman, Inc., Palo Alto, Calif. All solvents (exceptsTFA and isopropanol) were distilled before use. To check homogeneity thepeptides were chromatographed on precoated TLC plates (silica gel 60F-254 E. Merck, Darmstadt, Germany).

The peptides was built up by the stepwise solid-phase manual method onbenzhydrylamine hydrochloride resin. The procedure of synthesis was asfollows:

Deprotection. (1) CH₂ Cl₂ (wash 2 times, 2 min.); (2) 50% TFA in CH₂ Cl₂containing 0.1% indole (wash 1 time, 2 min.); (3) 50% TFA in CH₂ Cl₂containing 0.1% indole (deprotection, 30 min.); (4) CH₂ Cl₂ (wash 3times, 2 min.).

Neutralization. (1) CH₂ Cl₂ (wash 2 times, 2 min.); (2) 10% Et₃ N in CH₂Cl₂ (wash 1 time, 1 min.); (3) 10% Et₃ N in CH₂ Cl₂ (neutralization, 5min.); (4) CH₂ Cl₂ (wash 5 times, 2 min.).

DCC Coupling. (1) CH₂ Cl₂ (wash 2 times, 2 min.); (2) amino acid insolution in CH₂ Cl₂ was added (3) 10% DCC in CH₂ Cl₂ was added (at roomtemperature for 3-6 hr); (4) CH₂ Cl₂ (wash 2 times, 2 min.); (5) DMF(wash 3 times, 2 min.); (6) i-PrOH (wash 2 times, 2 min.); (7) CH₂ Cl₂(wash 2 times, 2 min.).

Active Ester Coupling. (1) CH₂ Cl₂ (wash 2 times, 2 min.); (2) aminoacid solution in DMF and a few mg of 1-hydroxybenzotriazole as acatalyst were added (at room temperature for 6-24 hr); (3) CH₂ Cl₂ (wash2 times, 2 min.); (4) DMF (wash 2 times, 2 min.); (5) 2-PrOH (wash 3times, 2 min.); (6) CH₂ Cl₂ (wash 3 times, 2 min.).

Acetylation. (1) CH₂ Cl₂ (wash 2 times, 2 min.); (2) 25% Ac₂ O and 25%pyridine in CH₂ Cl₂ (acetylation, 20 min.); (3) CH₂ Cl₂ (wash 4 times, 2min.).

The first amino acid BOC-Leu was attached to the resin 1 g (0.75 mM NH₂/g) by the following procedure: neutralization and DCC coupling. Beforeplacing the resin into the 30 ml Funnel Buchner (This funnel wasconnected with a 250 ml filter flask. The side tube of the filter flaskwas connected with a water pump only when it was needed for the washingsolvent. Whenever the Buchner funnel was used as a reaction container,the side tube of the filter flask was sealed by a rubber bulb forprotecting against leakage of the reaction solution). The resin waswashed twice in a separate funnel with 25 ml of CH₂ Cl₂ /g resin toremove the fine particles. In all couplings, a 4-fold excess of theprotected amino acid was used. This procedure generally resulted in acomplete coupling. If a positive ninhydrin color was observed, a secondcoupling was needed with the 4-fold excess of protected amino acid. Thenthe resin was acetylated. The next amino acid was attached by thefollowing procedure: deprotection, neutralization and DCC coupling oractive ester condensation. The volume of the solvents and reagents usedfor washing and performing chemical reaction was about 10 ml/g resin.The acetylation mixture was freshly prepared before each use.

Cleavage of Peptide from Resin. After all of the amino acids had beencoupled, the protected peptide resin was dried overnight, in vacuum, byan oil pump. The resin weighed 2.01 g. The resin containing 10%distilled anisole was treated with double-distilled and dried (overCoF₃) liquid hydrogen fluoride (about 25 ml) for 1 hr at 0° C. Then, theHF was evaporated under reduced pressure by a water pump, and theresidue was dried overnight, in vacuum, by an oil pump. The mixture wasthen extracted twice with EtOAc (70 ml) and then twice with 70 ml of 12%HOAc and twice with 15 ml water. The combined aqueous solution waslyophilized to obtain the crude peptide; 1100 ml.

Purification of the Peptide. 1100 mg of the crude peptide was applied onSephadex G-25 column (100×2.5 cm) which had been equilibrated with 12%HOAc. Elution was with the same solvent. Fractions of 10 ml werecollected. Each fraction was tested on silica gel plates with thesolvent system n-BuOH:HOAc:H₂ O=4:1:2. Fractions 36-42 were collected.These combined fractions were lyophilized; 210 mg of product sample wasobtained. The peptide was applied on silica gel column (100×1.5 cm),which had been equilibrated with a solvent mixture of n-BuOH:HOAc:H₂O=4:1:2, and then eluated with the same solvent system. Fractions of 3ml were collected. Fractions 105-120 were combined and lyophilized;yield, 100 mg.

Amino Acid Analysis. The automatic amino acid analysis were performed ona Beckman Model 119 Automatic amino acid analyzer. The peptide washydrolyzed for 24 hrs in a sealed glass tube at 110° C. in 6N HCl. Themixture was then dried, in vacuo. The residue was dissolved in 1.5 ml ofsodium citrate buffer, pH 2.2 and 0.2 ml of the solution was applied tothe analyzer.

Glu 1.976(2), Pro 2.039(2); Leu 1.986(2); Phe 0.990(1); Lys 1.003(1);Arg 1.000(1); NH₃ (+); Trp(+).

High Pressure Liquid Chromatography. HPLC was performed on a Watersliquid chromatograph equipped with a Waters 660 solvent programmer. Thesamples was chromatographed on a μ-Bondapak C₁₈ (5μ) column (3.9×300mm). For elution of the peptide, a linear gradient from 20-100% ofsolvent B in 25 min. was used (solvent A: 0.1 potassium phosphatebuffer, pH 3.0; solvent B; 30% solvent A, 70% CH₃ CN). The flow rate was2 ml/min., 10 μl of a 0.1% solution of the peptide was injected. Theeluted peptide was detected by its UV-absorbance at 206 nm. Theretention time of the peptide was 14.5 min. and the purity was 98%.

Optical Rotation. Optical rotation, [α]_(D), was measured at roomtemperature with a Perkin Elmer 141 polarimeter. The peptide wasdissolved in 12% HOAc (5 mg/ml). [α]_(D) =-67.8.

TLC. Thin layer chromatography in 5 solvent systems: (1) n-BuOH:HOAc:H₂O=4:1:2, Rf 0.25; (2) n-BuOH:Pry:HOAc:H₂ O=30:30:6:2:4, Rf 0.58; (3)n-BuOH:EtOAc:HOAc:H₂ O=2:2:1:1, Rf 0.01; (4) EtOAc:Pyr:HOAc:H₂O=5:5:1:3, Rf 0.59; (5) n-BuOH:Pyr:HOAc:H₂ O=50:30:1:40, Rf 0.54.

ABBREVIATIONS AND FORMULAS

n-BuOH: n-butyl alcohol

HOAc: acetic acid

CH₃ Cn: acetonitrile

K H₂ PO₄ : potassium phosphate

o-Cl-Z: o-chlorobenzyloxycarbonyl

CH₂ Cl₂ : dichloromethane

TFA: trifluoroacetic acid

Et₃ N: triethylamine

DCC: dicyclohexylcarbodiimide

DMF: dimethylformamide

i-PrOH: isopropyl alcohol

BOC: t-butyloxycarbonyl

COF₃ : cobalt (III) fluoride

HF: hydrogen fluoride

EtOAc: ethyl acetate

Characterization data on the analogs of substance P from which evolvedthe invention are in Tables I, II and III.

                                      TABLE I                                     __________________________________________________________________________    Characterization Data on the Analogs of Substance P                           Substance P: Arg--Pro--Lys--Pro--Gln--Gln--Phe--Phe--Gly--Leu--Met--NH.sub    .2                                                                                                        Rf in Solvent System*                             Analog                      I  II III                                                                              IV V                                     __________________________________________________________________________    I. [D-Phe.sup.5,D-Trp.sup.7,D-Trp.sup.9,Leu.sup.11 ]--SP                                                  0.56                                                                             0.81                                                                             0.86                                                                             0.81                                                                             0.92                                  II.                                                                              [D-Arg.sup.1,D-Pro.sup.2,D-Phe.sup.5,D-Trp.sup.7,D-Trp.sup.9,Leu.sup.11        ]--SP                   0.92                                                                             0.04                                                                             0.61                                                                             0.37                                                                             0.25                                  III.                                                                             [Glp.sup.5,D-Trp.sup.7,D-Trp.sup.9,Thr.sup.11 ]--SP.sub.5-11                                           0.91                                                                             0  0.53                                                                             0.25                                                                             0.17                                  IV.                                                                              [D-Arg.sup.1,D-Pro.sup.2,D-Trp.sup.7,D-Trp.sup.9,Thr.sup.11 ]--SP                                      0.93                                                                             0.01                                                                             0.52                                                                             0.28                                                                             0.22                                  V. [Glp.sup.5,D-Trp.sup.7,D-Trp.sup.9,Leu.sup.11 ]--SP.sub.5-11                                           0.93                                                                             0.84                                                                             0.87                                                                             0.67                                                                             0.76                                  VI.                                                                              [D-Arg.sup.1,D-Trp.sup.7,D-Trp.sup.9,Leu.sup.11 ]--SP                                                  0.58                                                                             0.01                                                                             0.59                                                                             0.54                                                                             0.25                                  VII.                                                                             [D-Arg.sup.1,D-Pro.sup.2,D-Trp.sup.7,D-Trp.sup.9,Leu.sup.11 ]--SP                                      0.82                                                                             0  0.54                                                                             0.28                                                                             0.20                                  IX.                                                                              [D-Trp.sup.7,D-Trp.sup.9,Leu.sup.11 ]--SP                                                              0.82                                                                             0  0.57                                                                             0.35                                                                             0.18                                  X. [D-Arg.sup.1,D-Trp.sup.7 ,D-Trp.sup.9,Ile.sup.11 ]--SP                                                 0.89                                                                             0  0.56                                                                             0.29                                                                             0.27                                  XI.                                                                              [D-Arg.sup.1,D-Trp.sup.7,D-Trp.sup.9,Nle.sup.11 ]--SP                                                  0.67                                                                             0  0.63                                                                             0.38                                                                             0.28                                  __________________________________________________________________________     *I, EtOAc:py:HOAc:H.sub.2 O (5:5:1:3); II, nBuOH:EtOAc:HOAc:H.sub.2 O         (2:2:1:1); III, nBuOH:py:HOAc:H.sub.2 O (30:30:6:24); IV,                     nBuOH:py:HOAc:H.sub.2 O (50:33:1:40); V, nBuOH:HOAc:H.sub.2 O (4:1:2)    

                  TABLE II                                                        ______________________________________                                        Characterization Data on the Analogs of Substance P                                   Retention time  % Purity                                              Analog  in HPLC (min.)  (HPLC) ca.                                                                              [α].sub.D                             ______________________________________                                        I       17.5            98        +38.4**                                     II      16              97        -27*                                        III     --              --        +52**                                       IV      14.5            96        +38.6*                                      V       --              --        --                                          VI      14.5            98        -67.8*                                      VII     14              96        -50.4*                                      VIII    16.5            95        -38.7*                                      IX      15              97        -36.7*                                      X       17.5            97        -61.8*                                      XI                                                                            ______________________________________                                         *C 0.5%, 15% HOAc                                                             **C 0.5%, HOAc                                                           

                                      TABLE III                                   __________________________________________________________________________    Characterization Data on the Analogs of Substance P                           Amino Acid Analytical Data                                                    __________________________________________________________________________    I.   Glu 2.11(2); Leu 1.98(2); Phe 0.98*1); Trp (+).                          II.  Glu 0.93(1); Pro 1.98(2); Leu 2.06(2); Phe 2.00(2); Lys 1.03(1);              Arg 1.00(1); NH.sub.3 (+); Trp (+).                                      III. Glu 2.20(2); Thr 1.11(1); Leu 1.03(1); Phe 0.97(1); NH.sub.3 (+);             Trp (+).                                                                 IV.  Glu 1.98(2); Pro 2.04(2); Thr 0.94(1); Leu 1.02(1); Phe 0.98(1);              Lys 1.13(1); Arg 0.97(1); NH.sub.3 (+); Trp (+).                         V.   Glu 2.06(2); Phe 0.95(1); Leu 2.00(2); Trp (+).                          VI.  Glu 1.98(2); Pro 2.04(2); Leu 1.99(2); Phe 0.99(1); Lys 1.00(1);              Arg 1.00(1); NH.sub.3 (+); Trp (+).                                      VII. Glu 1.99(2); Pro 2.04(2); Leu 2.07(2); Phe 0.98(1); Lys 0.96(1);              Arg 0.96(1); NH.sub.3 (+); Trp (+).                                      VIII.                                                                              Glu 1.96(2); Pro 2.12(2); Leu 2.01(2); Phe 0.95(1); Lys 1.00(1);              Arg 0.96(1); NH.sub.3 (+); Trp (+).                                      IX.  Glu 1.98(2); Pro 2.35(2); Leu 1.97(2); Phe 0.96(1); Lys 0.97(1);              Arg 0.93(1); NH.sub.3 (+); Trp (+).                                      X.   Glu 1.01(2); Pro 1.02(2); Ile 0.91(1); Leu 1.02(1); Phe 0.97(1);              Lys 1.03(1); Arg 1.02(1); NH.sub.3 (+); Trp (+).                         __________________________________________________________________________

CHEMICAL AND BIOLOGICAL RESULTS

The antagonistic activities of the analogs were evaluated using theterminal portion of the guinea pig ileum, as described by Yamaguchi etal. [Acta Chem. Scand. B33, 63 (1979)]. The biological assay data are inTable IV.

The sequence of substance P (SP) is expressed in the top of Table IV.The antagonistic activity is defined as the fold-increase in theconcentration of SP in the presence of the analog to give 50% of themaximal response which is elicited by SP alone. The sequences of theanalogs which were designed and synthesized are in Table IV. The elevenamino acids of SP are spaced across the top line to facilitate visualcomparison of the amino acids in SP with those in the analogs. Forexample, analog I is also spaced across the page to show the sequencerelationship to SP. On this basis, analog I isH-Arg¹,Pro²,Lys³,Pro⁴,D-Phe⁵,Gln⁶,D-Trp.sup.7,Phe⁸ D-Trp⁹,Leu¹⁰,Leu¹¹-NH₂, but on the basis of conventional peptide nomenclature, analog I isexpressed as [D-Phe⁵,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-SP₅₋₁₁.

The truncated heptapeptide I required a >200-fold increase at 10⁻⁴ inthe concentration of SP to allow 50% of the maximal response of SP. WhenD-Arg¹,D-Pro² - were substituted for Arg¹,Pro² - to give theundecapeptide II corresponding to I in positions 5-11, the activity wasdecreased, as revealed by the 64-fold and >200-fold increase,respectively.

The truncated heptapeptide III had a low antagonistic activity of 2-foldat 10⁻⁴, and the corresponding undecapeptide, IV, with D-Arg¹,D-Pro² -had an increased antagonistic activity of 5-fold at 10⁻⁴.

The truncated heptapeptide V required at 17-fold increase at 10⁻⁴ andthe undecapeptide IX with the same sequence in positions 1-4, as in SP,required a 34-fold increase at 10⁻⁴. Consequently, for this matchingpair, the undecapeptide was twice as potent as the matchingheptapeptide. When L-Arg¹ of the undecapeptide IX was changed to D-Arg¹,as in the undecapeptide VI, the antagonistic activity was significantlyincreased from 34-fold to 625-fold. When L-Pro² of the undecapeptide IXwas changed to D-Pro² in the undecapeptide VIII, the antagonisticactivity was increased two-fold from 34-fold to 70-fold at 10⁻⁴. WhenL-Arg¹ in the undecapeptide VIII was changed to D-Arg¹ as in theundecapeptide VII, the antagonistic activity was again increased from70-fold to 100-fold at 10⁻⁴. Alternatively, when L-Pro² of theundecapeptide VI was changed to D-Pro² in the undecapeptide VII, theantagonistic potency was decreased from 625-fold to 100-fold at 10⁻⁴.

Evaluation of the antagonistic potencies of the four undecapeptides,VI-IX, shows that it is significantly beneficial to have theD-configuration of Arg in position 1 of SP for an antagonist, but toretain the L-configurations of Pro²,Lys³,Pro⁴ in this N-terminalsequence of four amino acids in SP.

                                      TABLE IV                                    __________________________________________________________________________    Biological Assay Data for Antagonism                                                                                          Fold                                                                          increase                                                                      in conc.                      __________________________________________________________________________    (SP)                                                                              H--Arg.sup.1                                                                       Pro.sup.2                                                                         Lys.sup.3 Pro.sup.4                                                                Gln.sup.5                                                                         Gln.sup.6                                                                        Phe.sup.7                                                                         Phe.sup.8                                                                         Gly.sup.9                                                                        Leu.sup.10                                                                        Met.sup.11 NH.sub.2                   I.  [             D-Phe.sup.5                                                                          D-Trp.sup.7                                                                          D-Trp.sup.9                                                                           Leu.sup.11 ]--SP.sub.5-11                                                             200/10.sup.-4 *               II. [D-Arg.sup.1                                                                       D-Pro.sup.2                                                                            D-Phe.sup.5                                                                          D-Trp.sup.7                                                                          D-Trp.sup.9                                                                           Leu.sup.11 ]--SP                                                                      64/10.sup.-4                                                                   7/10.sup.-5                  III.                                                                              [             Glp.sup.5                                                                            D-Trp.sup.7                                                                          D-Trp.sup.9                                                                           Thr.sup.11 ]--SP.sub.5-11                                                              2/10.sup.-4                  IV. [D-Arg.sup.1                                                                       D-Pro.sup.2     D-Trp.sup.7                                                                          D-Trp.sup.9                                                                           Thr.sup.11 ]--SP                                                                      5×/10.sup.-4            V.  [             Glp.sup.5                                                                            D-Trp.sup.7                                                                          D-Trp.sup.9                                                                           Leu.sup.11 ]--SP.sub.5-                                                               17×/10.sup.-4           VI. [D-Arg.sup.1         D-Trp.sup.7                                                                          D-Trp.sup.9                                                                           Leu.sup.11 ]--SP                                                                      625/10.sup.-4                 VII.                                                                              [D-Arg.sup.1                                                                       D-Pro.sup.2     D-Trp.sup.7                                                                          D-Trp.sup.9                                                                           Leu.sup.11 ]--SP                                                                      100/10.sup.-4                                                                 17/10.sup.-5                  VIII.                                                                             [    D-Pro.sup.2     D-Trp.sup.7                                                                          D-Trp.sup.9                                                                           Leu.sup.11 ]--SP                                                                      70/10.sup.-4                                                                  13/10.sup.-5                  IX. [                    D-Trp.sup.7                                                                          D-Trp.sup.9                                                                           Leu.sup.11 ]--SP                                                                      34/10.sup.-4                  __________________________________________________________________________     *difficulty soluble in 50% ethanol.                                      

For two pairs of heptapeptides and undecapeptides having relatively lowantagonistic activities, the undecapeptides had somewhat higherantagonistic activities. When an undecapeptide of high antagonisticactivity (625-fold at 10⁻⁴) is compared with the correspondingheptapeptide, it is the undecapeptide which is significantly morepotent. Since it is the antagonist of high potency which is importantfor physiological research, the undecapeptide is more important than thetruncated heptapeptide.

These results indicate that the N-terminal Arg¹,Pro²,Lys³,Pro⁴ areapparently very important for binding at a receptor even though thesefour amino acids may not be directly involved in mechanisms in agonism.Although the heptapeptide has antagonistic activity, the use of theundecapeptide in physiology may better reveal intrinsic mechanisms,i.e., the heptapeptide might reveal more artifactual relationships.

The fact that Arg¹ has the D- rather than the natural configuration inVI indicates possible greater enzymic stability. The superiority ofD-Arg¹ over L-Arg¹ for both undecapeptides, VI and VII, is revealed bythe increases to 625-fold and 100-fold, respectively, at 10⁻⁴ incomparison with IX and VIII at 34-fold and 70-fold, respectively.

These pairs of heptapeptides and undecapeptides show that a heptapeptideis not necessarily as antagonistically potent as the correspondingundecapeptide, but rather that undecapeptides may be more active thanthe corresponding heptapeptides. The undecapeptide IX is more activethan the corresponding heptapeptide V by the potencies of 34-fold and17-fold, respectively.

Uses for antagonists of substance P may be multiple; for example, theuse of an antagonist for inflammatory responses in the human eye mightbe better with an undecapeptide, but uses of truncated antagonists mightbe fully satisfactory for analgesic action in an intact mammalianspecies.

These undecapeptides and truncated peptides which are based on thesequence of substance P, but which have antagonistic activity ratherthan agonistic activity, have several uses. The availability of such anantagonist like [D-Arg¹,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-Sp makes possible theelucidation of the mechanisms of multiply biological actions ofsubstance P, which is quite impossible in the absence of suchantagonists. Since substance P itself is apparently a neurogenicmediator of the inflammatory response in the human eye, this antagonistof substance P is useful in ophthalmology to therapeutically treatinflammatory responses in the human eye, and the therapeutic applicationmay be topical, which is very practical. Another use involves the actionof such an antagonist to block substance P in pain mechanisms, and onthis basis the antagonist has a useful analgesic action.

The several examples of antagonists of substance P set forth in thisdocument exemplify a category of analogs which have useful and potentantagonistic activity. It is obvious that other substituents in thepositions of the undecapeptides and truncated peptides, which areclearly based on the structures of the disclosed peptides, are entirelywithin the scope of this invention.

What is claimed:
 1. [D-Arg¹,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-Sp. 2.[D-Phe⁵,D-Trp⁷,D-Trp⁹,Leu¹¹ ]-SP_(5-1l).
 3. Substance P having aminoacid substitutions at one or more of positions 1, 2, 5, 7, 9, or 11,wherein the amino acid substitution at position 1 is D-Arg; position 2is D-Pro; position 5 is D-Phe; position 7 is D-Trp; position 9 is D-trp;and position 11 is Leu.
 4. The substituted Substance P of claim 3 whichis [D-Arg¹, D-Pro², D-Phe⁵, D-Trp⁷, D-Trp⁹, Leu¹¹ ]-SP.
 5. Thesubstituted Substance P of claim 3 which is [D-Arg¹, D-Pro², D-Trp⁷,D-Trp⁹, Leu¹¹ ]-SP.
 6. The substituted Substance P of claim 3 which is[D-Pro², D-Trp⁷, D-Trp⁹, Leu¹¹ ]-SP.
 7. The substituted Substance P ofclaim 3 which is [D-Trp⁷, D-Trp⁹, Leu¹¹ ]-SP.